Research on the mechanism of EGFR/Foxo3a/Snail1 pathway in bleomycin-induced pulmonary fibrosis in mice_Chinese Journal of Respiratory and Critical Care Medicine

Authors：

YANG Weiyuan ^1,2 , LI Li ² , ZHENG Linxin ² ,  LI Weifeng ²

1. Guangzhou University of Traditional Chinese Medicine, Guangzhou, Guangdong 510405, P. R. China;
2. General Hospital of Guangzhou Theatre Command, Guangzhou, Guangdong 510010, P. R. China;

Corresponding author：

LI Weifeng, Email: lwf980622@126.com

Keywords：

Forkhead box O3a; Pulmonary fibrosis; Epithelial mesenchymal transition; Bleomycin; Gefitinib

DOI：

10.7507/1671-6205.201904028

Video：

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Objective By intervening with gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor, to explore the downstream signaling pathway of the transcription factor forkhead box O3a (Foxo3a) in C57BL/6 mice who are induced to pulmonary fibrosis with bleomycin, as so to illuminate the possible mechanism of Foxo3a in epithelial-mesenchymal transition (EMT) of pulmonary fibrosis.Methods Thirty C57BL/6 mice aged 6 weeks in half genders were randomly divided into a control group, a bleomycin group and a gefitinib group. The mice in the control group were injected with saline via trachea. The mice in the bleomycin group were injected with bleomycin at a dose of 3 mg/kg via trachea. The mice in the gefitinib group were injected with bleomycin at a dose of 3 mg/kg via trachea and then gastrically perfused with gefitinib (20 mg·kg^–1·d^–1). 14 days after the treatment, all mice were killed and lung tissue specimens were collected for further detection. Lung tissue sections were stained with hematoxylin eosin and Masson’s trichrome. The mRNA levels of α-smooth muscle actin (α-SMA), E-cadherin, high mobility group protein box 1 (HMGB1), Foxo3a, FoxM1 and Snail1 in the lung tissues were detected by RT-PCR. The protein expressions of α-SMA, E-cadherin, HMGB1, phospho-Foxo3a (p-Foxo3a), Foxo3a, FoxM1 and Snail1 in the lung tissues were determined by western blot.Results The scores of lung inflammation and fibrosis were evidently decreased in the gefitinib group compared with that in the bleomycin group (P<0.01). Compared with bleomycin group, the mRNA level of α-SMA, Snail1 (P<0.01) and HMGB1 (P<0.05) were declined, but mRNA level of E-cadherin (P<0.01), Foxo3a and FoxM1 (P>0.05) were ascendant in the gefitinib group. Meanwhile, western blot analysis showed reduced protein expressions of α-SMA (P<0.05), Snail1(P<0.01), HMGB1 (P<0.05) and p-Foxo3a/Foxo3a (P<0.01) in lung tissues, while expressions of E-cadherin (P<0.05), Foxo3a and FoxM1 proteins (P>0.05) were increased in the gefitinib group.Conclusions Increased activity of Foxo3a can down-regulate Snail1, which decreases the expression of α-SMA and increases the expression of E-cadherin, thereby attenuating bleomycin-induced pulmonary fibrosis in mice.

Citation： YANG Weiyuan, LI Li, ZHENG Linxin, LI Weifeng. Research on the mechanism of EGFR/Foxo3a/Snail1 pathway in bleomycin-induced pulmonary fibrosis in mice. Chinese Journal of Respiratory and Critical Care Medicine, 2020, 19(4): 371-378. doi: 10.7507/1671-6205.201904028 Copy

1.	葛均波, 徐永健, 王辰, 主编. 内科学. 第 9 版. 北京: 人民卫生出版社, 2018, 87.
2.	Kim KK, Kugler MC, Wolters PJ, et al. Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proc Natl Acad Sci U S A, 2006, 103(35): 13180-13185.
3.	Margetts PJ, Bonniaud P, Liu L, et al. Transient overexpression of TGF-β1 induces epithelial mesenchymal transition in the rodent peritoneum. J Am Soc Nephrol, 2005, 16: 425-436.
4.	Fang L, Wang H, Zhou L, et al. Akt-FoxO3a signaling axis dysregulation in human oral squamous cell carcinoma and potent efficacy of FoxO3a-targeted gene therapy. Oral Oncol, 2011, 47(1): 16-21.
5.	蒋宗胜. 基于转录因子 FOXM1 调控下的抗非小细胞肺癌上皮间质转化的机制研究[D]. 2018, 福建医科大学. 48-49.
6.	Ishii Y, Fujimoto S, Fukuda T. Gefitinib prevents bleomycin-induced lung fibrosis in mice. Am J Resp Crit Care Med, 2006, 174: 550-556.
7.	Le Cras TD, Korfhagen TR, Davidson C, et al. Inhibition of PI3K by PX-866 prevents transforming growth factor-alpha-induced pulmonary fibrosis. Am J Pathol, 2010, 176(2): 679-686.
8.	Wang XT, McCullough KD, TF Franke, et al. Epidermal growth factor receptor-dependent Akt activation by oxidative stress enhances cell survival. J Biol Chem, 2000, 275(19): 14624-14631.
9.	Gomes AR, Zhao F, Lam EWF. Role and regulation of the forkhead transcription factors FOXO3a and FOXM1 in carcinogenesis and drug resistance. Chin J Cancer, 2013, 32(7): 365-370.
10.	李伟峰, 李理, 袁伟锋, 等. 吉非替尼抑制博莱霉素诱导的小鼠肺纤维化. 中国病理生理杂志, 2010, 26(8): 1565-1569.
11.	李理, 李伟峰, 蔡琳, 等. 吉非替尼对博莱霉素致肺纤维化小鼠 α 平滑肌肌动蛋白的抑制作用. 南方医科大学学报, 2010, 30(12): 2675-2678.
12.	彭婷, 杜海坚, 李理, 等. 吉非替尼通过增加 Foxo3a 活性抑制肺纤维化小鼠上皮–间质转分化. 中国病理生理杂志, 2014, 30(3): 444-448.
13.	熊俊. 肾间质纤维化中的上皮–间充质转型及 Snail1 的作用研究[D]. 2011, 第二军医大学. 12.
14.	Mikawa K, Nishina K, Takao Y, et al. ONO-1714, a nitric oxide synthase inhibitor, attenuates endotoxin-induced acute lung injury in rabbits. Anesth Analg, 2003, 97(6): 1751-1755.
15.	Ashcroft T, Simpson JM, Timbrell V. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol, 1998, 41(4): 467-470.
16.	赵永强, 楚海燕, 膝礼, 等. 博莱霉素诱导肺纤维化小鼠模型的建立及特征. 疑难病杂志, 2013, 12(9): 705-709.
17.	李伟峰, 胡玉洁, 袁伟锋, 等. 气管内滴入与雾化博莱霉素致小鼠肺纤维化模型的比较研究. 南方医科大学学报, 2012, 32(2): 221-225.
18.	齐曼古丽吾守尔, 夏宇, 巴哈尔古丽米吉提, 等. 博莱霉素致大鼠肺纤维化模型的建立方法及比较. 新疆医科大学学报, 2005, 28(6): 495-498.
19.	刘祉宁. 上皮–间充质转化与肺纤维化的研究进展. 齐齐哈尔医学院学报, 2018, 39(8): 950-953.
20.	胡青青, 周恩敏, 金克, 等. 褪黑素对博来霉素诱导肺纤维化小鼠 α-SMA 和 E-cadherin 表达的影响. 温州医科大学学报, 2018, 48(8): 577-581.
21.	蒋宗胜, 阮君山, 王少明. FOX 家族介导的上皮间质转化在肿瘤转移中的研究进展. 中国药理学通报, 2016, 32(8): 1053-1058.
22.	张郡, 李长天, 赵腾蛟. FOXM1、FOXO3a 在肺腺癌中的表达及临床意义. 甘肃科技, 2018, 34(5): 124-127.
23.	McGovern UB, Francis RE, Peck B, et al. Gefitinib (Iressa) represses FOXM1 expression via FOXO3a in breast cancer. Mol Cancer Ther, 2009, 8(3): 582-591.
24.	张均. Snail 及 α-SMA 在 IgA 肾病中的表达及意义[D]. 2015, 重庆医科大学. 21.
25.	蔡琳, 李理, 徐军. 高迁移率族蛋白 B1 和 α 平滑肌肌动蛋白在博莱霉素诱导肺纤维化中的表达. 中国呼吸与危重监护杂志, 2009, 8(2): 181-185.
26.	郑林鑫, 陈灿, 麦玉梅, 等. 两种表皮生长因子抑制剂下调肿瘤抑制素 M 抑制博来霉素诱导的小鼠肺纤维化. 中国呼吸与危重监护杂志, 2018, 17(4): 400-406.
27.	吴海兰, 戴伟, 赵明, 等. 高迁移率族蛋白 B1 在急性肺损伤中的研究进展. 临床肺科杂志, 2019, 24(12): 2276-2280.
28.	Voutsadakis IA. The ubiquitin-proteasome system and signal transduction pathways regulating epithelial mesenchymal transition of cancer. J Biomed Sci, 2012, 19(1): 67.
29.	Nho RS, Hergert P, Kahm J, et al. Pathological alteration of FoxO3a activity promotes idiopathic pulmonary fibrosis fibroblast proliferation on type I collagen matrix. Am J Pathol, 2011, 179(5): 2420-2430.
30.	姚怡, 张建林, 赵虹, 等. 叉头框蛋白 O3a 表达下调对人肾小管上皮细胞 EMT 的影响. 中国医学创新, 2017, 12(13): 33-36.
31.	刘伏友, 孙岩, 孙林, 等. 去甲斑蝥素对 TGF-β1 诱导的人近端肾小管细胞上皮间质转分化以及转录因子 Snail1 表达的影响. 中国中西医结合肾病杂志, 2009, 10(11): 952-957.
32.	Balli D, Ustiyan V, Zhang YF, et al. FoxM1 transcription factor is required for lung fibrosis and epithelial-to-mesenchymal transition. EMBO J, 2013, 32(2): 231-244.
33.	金成, 方钿德, 詹杰锋, 等. FoxM1 与 EMT 相关蛋白 E-Cadherin 在结肠癌中的表达及其相关性研究. 实用预防医学, 2018, 25(5): 594-596.
34.	于水, 于春鹏, 石磊, 等. FoxM1 调控 Snail1 促进肝癌上皮–间充质转换的作用. 青岛大学医学院学报, 2016, 52(6): 645-654.
35.	Ye H, Kelly TF, Samadani U, et al. Hepatocyte nuclear factor 3/Forkhead homolog 11 is expressed in proliferating epithelial and mesenchymal cells of embryonic and adult tissues. Mol Cell Biol, 1997, 17(3): 1626-1641.
36.	Korver W, Roose J, Wilson A, et al. The winged-helix transcription factor trident is expressed in cycling cells. Nucleic Acids Res, 1997, 25(9): 1715-1724.
37.	谭拥军, 陈含笑. 转录因子 FoxM1 剪接异构体在乳腺癌 EMT 过程中的初步研究. 湖南大学学报, 2015, 42(12): 100-106.

1. 葛均波, 徐永健, 王辰, 主编. 内科学. 第 9 版. 北京: 人民卫生出版社, 2018, 87.
2. Kim KK, Kugler MC, Wolters PJ, et al. Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proc Natl Acad Sci U S A, 2006, 103(35): 13180-13185.
3. Margetts PJ, Bonniaud P, Liu L, et al. Transient overexpression of TGF-β1 induces epithelial mesenchymal transition in the rodent peritoneum. J Am Soc Nephrol, 2005, 16: 425-436.
4. Fang L, Wang H, Zhou L, et al. Akt-FoxO3a signaling axis dysregulation in human oral squamous cell carcinoma and potent efficacy of FoxO3a-targeted gene therapy. Oral Oncol, 2011, 47(1): 16-21.
5. 蒋宗胜. 基于转录因子 FOXM1 调控下的抗非小细胞肺癌上皮间质转化的机制研究[D]. 2018, 福建医科大学. 48-49.
6. Ishii Y, Fujimoto S, Fukuda T. Gefitinib prevents bleomycin-induced lung fibrosis in mice. Am J Resp Crit Care Med, 2006, 174: 550-556.
7. Le Cras TD, Korfhagen TR, Davidson C, et al. Inhibition of PI3K by PX-866 prevents transforming growth factor-alpha-induced pulmonary fibrosis. Am J Pathol, 2010, 176(2): 679-686.
8. Wang XT, McCullough KD, TF Franke, et al. Epidermal growth factor receptor-dependent Akt activation by oxidative stress enhances cell survival. J Biol Chem, 2000, 275(19): 14624-14631.
9. Gomes AR, Zhao F, Lam EWF. Role and regulation of the forkhead transcription factors FOXO3a and FOXM1 in carcinogenesis and drug resistance. Chin J Cancer, 2013, 32(7): 365-370.
10. 李伟峰, 李理, 袁伟锋, 等. 吉非替尼抑制博莱霉素诱导的小鼠肺纤维化. 中国病理生理杂志, 2010, 26(8): 1565-1569.
11. 李理, 李伟峰, 蔡琳, 等. 吉非替尼对博莱霉素致肺纤维化小鼠 α 平滑肌肌动蛋白的抑制作用. 南方医科大学学报, 2010, 30(12): 2675-2678.
12. 彭婷, 杜海坚, 李理, 等. 吉非替尼通过增加 Foxo3a 活性抑制肺纤维化小鼠上皮–间质转分化. 中国病理生理杂志, 2014, 30(3): 444-448.
13. 熊俊. 肾间质纤维化中的上皮–间充质转型及 Snail1 的作用研究[D]. 2011, 第二军医大学. 12.
14. Mikawa K, Nishina K, Takao Y, et al. ONO-1714, a nitric oxide synthase inhibitor, attenuates endotoxin-induced acute lung injury in rabbits. Anesth Analg, 2003, 97(6): 1751-1755.
15. Ashcroft T, Simpson JM, Timbrell V. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol, 1998, 41(4): 467-470.
16. 赵永强, 楚海燕, 膝礼, 等. 博莱霉素诱导肺纤维化小鼠模型的建立及特征. 疑难病杂志, 2013, 12(9): 705-709.
17. 李伟峰, 胡玉洁, 袁伟锋, 等. 气管内滴入与雾化博莱霉素致小鼠肺纤维化模型的比较研究. 南方医科大学学报, 2012, 32(2): 221-225.
18. 齐曼古丽吾守尔, 夏宇, 巴哈尔古丽米吉提, 等. 博莱霉素致大鼠肺纤维化模型的建立方法及比较. 新疆医科大学学报, 2005, 28(6): 495-498.
19. 刘祉宁. 上皮–间充质转化与肺纤维化的研究进展. 齐齐哈尔医学院学报, 2018, 39(8): 950-953.
20. 胡青青, 周恩敏, 金克, 等. 褪黑素对博来霉素诱导肺纤维化小鼠 α-SMA 和 E-cadherin 表达的影响. 温州医科大学学报, 2018, 48(8): 577-581.
21. 蒋宗胜, 阮君山, 王少明. FOX 家族介导的上皮间质转化在肿瘤转移中的研究进展. 中国药理学通报, 2016, 32(8): 1053-1058.
22. 张郡, 李长天, 赵腾蛟. FOXM1、FOXO3a 在肺腺癌中的表达及临床意义. 甘肃科技, 2018, 34(5): 124-127.
23. McGovern UB, Francis RE, Peck B, et al. Gefitinib (Iressa) represses FOXM1 expression via FOXO3a in breast cancer. Mol Cancer Ther, 2009, 8(3): 582-591.
24. 张均. Snail 及 α-SMA 在 IgA 肾病中的表达及意义[D]. 2015, 重庆医科大学. 21.
25. 蔡琳, 李理, 徐军. 高迁移率族蛋白 B1 和 α 平滑肌肌动蛋白在博莱霉素诱导肺纤维化中的表达. 中国呼吸与危重监护杂志, 2009, 8(2): 181-185.
26. 郑林鑫, 陈灿, 麦玉梅, 等. 两种表皮生长因子抑制剂下调肿瘤抑制素 M 抑制博来霉素诱导的小鼠肺纤维化. 中国呼吸与危重监护杂志, 2018, 17(4): 400-406.
27. 吴海兰, 戴伟, 赵明, 等. 高迁移率族蛋白 B1 在急性肺损伤中的研究进展. 临床肺科杂志, 2019, 24(12): 2276-2280.
28. Voutsadakis IA. The ubiquitin-proteasome system and signal transduction pathways regulating epithelial mesenchymal transition of cancer. J Biomed Sci, 2012, 19(1): 67.
29. Nho RS, Hergert P, Kahm J, et al. Pathological alteration of FoxO3a activity promotes idiopathic pulmonary fibrosis fibroblast proliferation on type I collagen matrix. Am J Pathol, 2011, 179(5): 2420-2430.
30. 姚怡, 张建林, 赵虹, 等. 叉头框蛋白 O3a 表达下调对人肾小管上皮细胞 EMT 的影响. 中国医学创新, 2017, 12(13): 33-36.
31. 刘伏友, 孙岩, 孙林, 等. 去甲斑蝥素对 TGF-β1 诱导的人近端肾小管细胞上皮间质转分化以及转录因子 Snail1 表达的影响. 中国中西医结合肾病杂志, 2009, 10(11): 952-957.
32. Balli D, Ustiyan V, Zhang YF, et al. FoxM1 transcription factor is required for lung fibrosis and epithelial-to-mesenchymal transition. EMBO J, 2013, 32(2): 231-244.
33. 金成, 方钿德, 詹杰锋, 等. FoxM1 与 EMT 相关蛋白 E-Cadherin 在结肠癌中的表达及其相关性研究. 实用预防医学, 2018, 25(5): 594-596.
34. 于水, 于春鹏, 石磊, 等. FoxM1 调控 Snail1 促进肝癌上皮–间充质转换的作用. 青岛大学医学院学报, 2016, 52(6): 645-654.
35. Ye H, Kelly TF, Samadani U, et al. Hepatocyte nuclear factor 3/Forkhead homolog 11 is expressed in proliferating epithelial and mesenchymal cells of embryonic and adult tissues. Mol Cell Biol, 1997, 17(3): 1626-1641.
36. Korver W, Roose J, Wilson A, et al. The winged-helix transcription factor trident is expressed in cycling cells. Nucleic Acids Res, 1997, 25(9): 1715-1724.
37. 谭拥军, 陈含笑. 转录因子 FoxM1 剪接异构体在乳腺癌 EMT 过程中的初步研究. 湖南大学学报, 2015, 42(12): 100-106.

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Research on the mechanism of EGFR/Foxo3a/Snail1 pathway in bleomycin-induced pulmonary fibrosis in mice

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